Reverse transcriptases are foundational enzymes in biotechnology that convert RNA into DNA. These enzymes form the basis of valuable research tools that have been used to uncover many of the fundamental processes of living organisms. With respect to molecular diagnostics, these enzymes are critical components of such diagnostics, thus facilitating new tools for the diagnosis and management of the vast majority of diseases, including cancer for example. As such, improved reverse transcriptases with improved properties, such as improved efficiency, are desirable, since such improved enzymes will lead to improved molecular diagnostics.
A factor that influences the efficiency of reverse transcription is the ability of RNA to form secondary structures. Such secondary structures can form, for example, when regions of RNA molecules have sufficient complementarity to hybridize and form double stranded RNA. Generally, the formation of RNA secondary structures can be reduced by raising the temperature of solutions which contain the RNA molecules. Thus, in many instances, it is desirable to reverse transcribe RNA at temperatures above 37° C. However, reverse transcriptases generally lose activity when incubated at temperatures much above 37° C. (e.g., 50° C.).
The accuracy of methods utilizing reverse transcriptases, including molecular diagnostics methods using such enzymes, would be improved by the discovery of reverse transcriptases with improved thermostability and/or thermoreactivity. If such enzymes were available, then methods employed for other thermostable enzymes to improve accuracy, could be used to conceive new methods utilizing thermostable reverse transcriptases. For instance, ‘hot start’ approaches have been employed with thermostable polymerases to improve the accuracy of polymerase chain reaction (PCR) methods. In one example, U.S. Pat. No. 5,338,671 describes the use of antibodies specific for a thermostable DNA polymerase to inhibit the DNA polymerase activity at low temperatures (e.g. <70° C.). Chemical treatment with citraconic anhydride is another way hot start PCR has been achieved (see, e.g., U.S. Pat. No. 5,773,258 and U.S. Pat. No. 5,677,152). The application of such hot start approaches to reverse transcription has proven to be challenging. This is because, for example, many reverse transcriptases are not heat-stable.
Moreover, biological samples from which nucleic acids are extracted often contain additional compounds that are inhibitory to reverse transcription. Humic acid in soil, plants and feces, hematin in blood, immunoglobin G in serum, and various blood anticoagulants, like heparin and citrate, are all examples of such inhibitors. Such inhibitors may not be completely removed during the nucleic acid extraction and purification process, thus negatively impacting downstream nucleic acid synthesis, as reflected by a decrease in cDNA product produced as a result of reverse transcription.
Thus, improved reverse transcriptases, and compositions, kits and methods that include such reverse transcriptases which overcome some of the drawbacks mentioned above are met by the present invention.